PT  - JOURNAL ARTICLE
AU  - Sara C Humphreys
AU  - Mai B Thayer
AU  - Julie M Lade
AU  - Bin Wu
AU  - Kelvin Sham
AU  - Babak Basiri
AU  - Yue Hao
AU  - Xin Huang
AU  - Richard Smith
AU  - Brooke M Rock
TI  - Plasma and liver protein binding of GalNAc conjugated siRNA
AID  - 10.1124/dmd.119.086967
DP  - 2019 Jan 01
TA  - Drug Metabolism and Disposition
PG  - dmd.119.086967
4099  - http://dmd.aspetjournals.org/content/early/2019/05/16/dmd.119.086967.short
4100  - http://dmd.aspetjournals.org/content/early/2019/05/16/dmd.119.086967.full
AB  - Understanding siRNA fraction unbound (fu) in relevant physiological compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of GalNAc-conjugated siRNA using classical small molecule in vitro techniques, we observed that hydrodynamic radius was critical in determining the size exclusion limit requirements for fu isolation; largely validating the siRNA 'rigid rod' hypothesis and providing insight into size-based kidney and lymphatic filtration of these molecules. With this knowledge, we developed an orthogonally-validated 50 kDa MWCO ultrafiltration assay to quantify fu in biological matrices including human, non-human primate, rat, mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA fu,plasma and found that chemical modifications can modulate plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins.SIGNIFICANCE STATEMENT N/A